Transcript Molecular Spectroscopy talk_Meng Zhang.pptx

Study of Proton Transfer in
Escherichia coli Photolyase
Meng Zhang
Biophysics Graduate Program
The Ohio State University
June 21, 2013
Introduction
• Photolyase: Photoenzyme that utilizes blue light to repair UVdamaged DNA which causes skin cancer.
• Flavin cofactor: The catalytic cofactor of photolyase, flavin adenine
dinucleotide (FAD), has five redox states.
UV-vis spectroscopic properties of wild type Photolyase
• The four redox states: FAD, FAD⦁̶ , FADH⦁, and FADH̶ have different
UV-vis absorption spectra.
Redox conversions in EcPL mutants
• Site-directed mutagenesis
• N378C (N5 mutant)
• E363L
• N378C/E363L
• In the presence of substrate,
N378C/E363L can stabilize at
the FAD⦁̶ state under
continuous white light for
hours.
 Proton channels are
eliminated completely.
are
The proposed proton channel in Photolyase
• The proton relay channel through E363 and surface water
to N378 and then FAD;
• The proton diffusion channel through the substrate binding
pocket.
Reaction kinetics
k12
k23
k34
•⦁
FADox ⇌ FAD ⇌ FADH ⇌ FADHk21
k32
k43
N378C with
substrate
WT
WT
N378C
Reaction kinetics
k12
k21
k23
k32
k34
k43
k43 with O2
5.8×10-4
0.15
0.31
3.3×10-4
0.013
<1.0×10-4
0.025
pH9
0.33
0.71
4.1×10-3
0.077
<1.0×10-4
8.2×10-3
pH7 with S
<1.0×10-4
0.19
<1.0×10-4
2.5×10-4
pH9 with S
<1.0×10-4
0.37
<1.0×10-4
5.6×10-3
0.088
0.050
>10
>10
<1.0×10-3
0.023
pH9
0.054
0.034
>10
>10
<1.0×10-4
0.046
pH7 with S
8.3×10-4
0.010
>10
<0.01
pH9 with S
2.2×10-4
6.3×10-4
>10
<0.01
0.015
0.018
<1.0×10-4
>1
pH9
0.021
0.032
<1.0×10-4
>1
pH7 with S
<1.0×10-4
0.031
<1.0×10-4
0.012
pH7
0.026
4.4×10-3
>10
>10
pH7
WT
pH7
N378C
pH7
E363L
N378C
E363L
k34
k12
k23
FADox ⇌ FAD•- ⇌ FADH⦁ ⇌ FADHk21
k32
k43
All in unit of min-1
6.7×10-4
9.7×10-4
7.3×10-3
Proton transfer in (6-4) Photolyase and Cryptochromes
• (6-4) Photolyase
•
•
•
Repairs (6-4) dipyrimidine
photoproducts.
Similar proton transfer
pathway.
Mutant N402C/E391L can
stabilizes at FAD⦁̶ state in the
presence of substrate.
• Cryptochromes (ongoing work)
FAD
PHR domain
C-terminal domain
• Insect Cryptochrome
• Plant Cryptochrome
Conclusions
• N378 and E363 are the key residues for EcPL proton transfer.
• The proton channels in photolyase was proposed for the first time. Proton
can be transported to flavin through two pathways: the proton relay channel
(through E363 and N378 in EcPL); and the proton diffusion channel through
the substrate binding pocket.
• Redox potentials of different FAD states are governed by the N5 residue
and local environment.
• The proton transfer kinetics are critical to the mechanistic and functional
divergence of Photolyase and Cryptochrome.
Acknowledgements
Prof. Dongping Zhong
Prof. Aziz Sancar (UNC)
Ms. Lijuan Wang
Dr. Jiang Li
Zheyun Liu
Chuang Tan
And all other colleagues in
the Zhong group!
Funding from:
NIH
Thank you!